This grant is directed at understanding the molecular mechanisms that control the development and connectivity of motor neurons. Since these cells are needed to control movement and respiration, diseases of motor neurons (e.g. ALS and SMA) are extremely costly and frequently lethal due to the lack of any treatment. The two main goals of this application are to characterize the function and biochemistry of motor neuron transcription factors and to identify the genetic pathways involved in their proper development. Our past studies have shown that LIM-HD factors function in a combinatorial manner to specify individual motor neuron subtypes (LIM code). In this grant we will examine how LIM-HD factors acquire cell type specific activities through functional and genetic interactions with other transcription factors. We will test whether LIM-HD factors have temporally regulated functions that direct the sequential refinement of motor neuron identity and function. Finally, we will use "forward" mouse genetic screens to identify and characterize new genes involved in motor neuron development. The experiments in this grant rely extensively on mouse genetics using transgenic and knockout methods, biochemistry and transcription assays, explant assays and imaging, and ENU-based mutagenesis screens. In aim one we will examine the function of LIM-HD factors Isl1 and Isl2, LMO factor LMO4, and Tbx factor Tbx20 using mouse knockout mutations to define functional interactions between LIM-HD factors and other classes of transcription factors expressed by motor neurons. In aim two we will use biochemical assays to investigate how gene regulation is controlled during motor neuron differentiation. In aim three we will examine whether motor neuron transcription factors Isl1, LMO4, Hb9, and Tbx20 are required for the survival and proper function of post natal motor neurons, since this could shed new light on motor neuron disease pathways. In aim four we will characterize new genetic pathways involved in motor neuron development by characterizing genes identified through an END mutagenesis screen. Our studies should provide novel information about the molecular pathways that operate to control motor neuron specification, axon navigation, circuit formation, and survival in adults.